Drought stress has a negative impact on plant growth and production. Arbuscular mycorrhizal (AM) fungi, which establish symbioses with most terrestrial vascular plant species, play important roles in improving host plant mineral nutrient acquisition and resistance to drought. However, the physiological and molecular regulation mechanisms occurring in mycorrhizal Eucalyptus grandis coping with drought stress remain unclear. Here, we studied the physiological changes and mitogen-activated protein kinase (MAPK) cascade gene expression profiles of E. grandis associated with AM fungi under drought stress. The results showed that colonization by AM fungi significantly enhanced plant growth, with higher plant biomass, shoot height, root length, and relative water content (RWC) under drought conditions. Mycorrhizal plants had lower levels of accumulation of proline, malondialdehyde (MDA), H2O2, and O2·- than seedlings not colonized with AM fungi. In addition, mycorrhizal E. grandis also had higher peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities under drought conditions, improving the antioxidant system response. Eighteen MAPK cascade genes were isolated from E. grandis, and the expression levels of the MAPK cascade genes were positively induced by symbiosis with AM fungi, which was correlated with changes in the proline, MDA, H2O2, and O2·- contents and POD, SOD, and CAT activities. In summary, our results showed that AM symbiosis enhances E. grandis drought tolerance by regulating plant antioxidation abilities and MAPK cascade gene expression. IMPORTANCE Arbuscular mycorrhizal (AM) fungi play an important role in improving plant growth and development under drought stress. The MAPK cascade may regulate many physiological and biochemical processes in plants in response to drought stress. Previous studies have shown that there is a complex regulatory network between the plant MAPK cascade and drought stress. However, the relationship between the E. grandis MAPK cascade and AM symbiosis in coping with drought remains to be investigated. Our results suggest that AM fungi could improve plant drought tolerance mainly by improving the antioxidant ability to protect plants from reactive oxygen species (ROS) and alleviate oxidative stress damage. The expression of the MAPK cascade genes was induced in mycorrhizal E. grandis seedlings under drought stress. This study revealed that MAPK cascade regulation is of special significance for improving the drought tolerance of E. grandis. This study provides a reference for improving mycorrhizal seedling cultivation under stress.

The majority of vascular flowering plants can establish arbuscular mycorrhizal (AM) symbiosis with AM fungi. These associations contribute to plant health and plant growth against various environmental stresses. In the mutualistic endosymbiosis, the AM fungi deliver phosphate (Pi) to the host root through highly branched hyphae called arbuscules. The molecular mechanisms of Pi transfer from AM fungi to the plant have been determined, which are dominated by AM-specific Pi transporters belonging to the PHOSPHATE TRANSPORTER 1 (Pht1) family within the subfamily I. However, it is unknown whether Pht1 family proteins are involved in other regulations in AM symbiosis. Here, we report that the expression of EgPT8 is specifically activated by AM fungus Rhizophagus irregularis and is localized in root cortical cells containing arbuscules. Interestingly, knockdown of EgPT8 function does not affect the Eucalyptus grandis growth, total phosphorous (P) concentration, and arbuscule formation; however, the size of mature arbuscules was significantly suppressed in the RNAi-EgPT8 lines. Heterogeneous expression of EgPT4, EgPT5, and EgPT8 in the Medicago truncatula mutant mtpt4-2 indicates that EgPT4 and EgPT5 can fully complement the defects of mutant mtpt4-2 in mycorrhizal Pi uptake and arbuscule formation, while EgPT8 cannot complement the defective AM phenotype of the mtpt4-2 mutant. Based on our results, we propose that the AM fungi-specific subfamily I transporter EgPT8 has novel functions and is essential to arbuscule elongation. IMPORTANCE Arbuscular mycorrhizal (AM) formation in host root cortical cells is initiated by exchanges of diffusible molecules, among which Pi uptake is known as the important feature of AM fungi on symbiosis functioning. Over the last two decades, it has been repeatedly proven that most vascular plants harbor two or more AM-specific Pht1 proteins; however, there is no direct evidence regarding the potential link among these Pi transporters at the symbiotic interface. This work revealed a novel function of a structurally conserved protein involved in lateral arbuscule development. In total, we confirmed that three AM-specific Pht1 family proteins are nonredundant in Eucalyptus grandis and that EgPT8 is responsible for fungal arbuscule elongation of Rhizophagus irregularis.